Accurate transmission of genetic information is important in the survival of a cell, an organism, and a species. A number of mechanisms have evolved that help to ensure high fidelity transmission of genetic material from one generation to the next since mutations can lead to new genotypes that may be deleterious to the cell. DNA lesions that frequently lead to mutations are modified, missing or mismatched nucleotides. Multiple enzymatic pathways have been described in prokaryotic systems that can specifically repair these lesions.
There are at least three ways in which mismatched nucleotides arise in DNA. First, physical damage to the DNA or DNA precursors can give rise to mismatched bases in DNA. For example, the deamination of 5-methyl-cytosine creates a thymine and, therefore, a G-T mispair. Second, misincorporation, insertion, or deletion of nucleotides during DNA replication can yield mismatched base pairs. Finally, genetic recombination produces regions of heteroduplex DNA which may contain mismatched nucleotides when such heteroduplexes result from the pairing of two different parental DNA sequences. Mismatched nucleotides produced by each of these mechanisms are known to be repaired by specific enzyme systems.
The well defined mismatch repair pathway is the E. coli MutHLS pathway that promotes a long-patch (approximately 3 Kb) excision repair reaction which is dependent on the mutH, mutL, mutS and MutU(uvrD) gene products. The MutHLS pathway appears to be the most active mismatch repair pathway in E. coli and is known to both increase the fidelity of DNA replication and act on recombination intermediates containing mispaired bases. This system has been reconstituted in vitro and requires the MutH, MutL, MutS and UvrD (helicase II) proteins along with DNA polymerase III holoenzyme, DNA ligase, single-stranded DNA binding protein (SSB) and one of the single-stranded DNA exonucleases, Exo I, Exo VII or RecJ. MutS protein binds to the mismatched nucleotides in DNA. MutH protein interacts with GATC sites in DNA that are hemi-methylated on the A and is responsible for incision on the unmethylated strand. Specific excision of the unmethylated strand results in increased fidelity of replication because excision is targeted to the newly replicated unmethylated DNA strand. MutL facilitates the interaction between MutS bound to the mismatch and MutH bound to the hemi-methylated Dam site resulting in the activation of MutH. UvrD is the helicase that appears to act in conjunction with one of the single-stranded DNA specific exonucleases to excise the unmethylated strand leaving a gap which is repaired by the action of DNA polymerase III holoenzyme, SSB and DNA ligase. In addition, E. coli contains several short patch repair pathways including the VSP system and the MutY (MicA) system that act on specific single base mispairs.
In bacteria, therefore, mismatch repair plays a role in maintaining the genetic stability of DNA. The bacterial MutHLS system has been found to prevent genetic recombination between the divergent DNA sequences of related species such as E. coli and S. typhimurium (termed: homeologous recombination).
The existence of prokaryotic mismatch repair systems that function to maintain genetic DNA stability is of particular interest since different types of human tumors show an instability of repeated DNA sequences. For example, Hereditary Non-Polyposis Colon Cancer (HNPCC), a familiar form of human colorectal cancer (CRC) that is also known as Lynch's Syndrome appears to be linked to a locus causing such genetic instability.
CRC is one of the most common forms of neoplasia in industrial countries and the possibility of a heritable component to CRC has been much debated. A high incidence of CRC within families has been well documented (approximately 13% of CRC cases are categorized as familial), but there is uncertainty over whether this effect results from common exposure to environmental influences such as diet, which have been shown to play a role in CRC risk, or from the influence of a genetic factor(s).
Recently, genetic linkage has been demonstrated between anonymous microsatellite markers on human chromosome 2 and the incidence of HNPCC. HNPCC is defined by the existence of at least three family members with CRC in at least two successive generations, with at least one affected member having been diagnosed at less than 50 years of age. A study of two independent HNPCC kindreds demonstrated the linkage with chromosome 2 markers, firmly supporting the view that there is a genetic component to HNPCC and suggesting that an unknown gene on chromosome 2 can play a role in conferring HNPCC susceptibility (Peltomaki et al., Science 260: 810, 1993, the contents of which are incorporated herein by reference). A further study of 14 smaller HNPCC kindreds also suggested a link between HNPCC and a gene on chromosome 2, although in this second study, the incidence of disease was not linked to markers on chromosome 2 in all families (Aaltonen et al. Science 260: 812, 1993).
Molecular analyses of HNPCC tumors have provided some information about likely characteristics of a gene responsible for conferring susceptibility to HNPCC. In particular, studies have revealed genomic instability of short repeated DNA sequences in HNPCC tumor tissues (Aaltonen et al., id; Thibodeau et al., Science 260: 816, 1993). The data also suggest that this tendency toward genomic instability can be inherited and may be related to mutation in a gene located on human chromosome 2. The idea that the mutation responsible for a genetic predisposition to HNPCC also leads to genomic instability of short repeated sequences is consistent with the observation that members of HNPCC kindreds show susceptibility to other cancers as well and often develop tumors outside the colorectal epithelium (e.g. in breast, ovary, bladder, endometrial (uterine), renal, skin or rectal). A full understanding of the relationship between mutation, genomic instability, and tumor development requires that the relevant genes be cloned and sequenced.
The problem is that cloning of genes involved in cancer development has proven difficult. In HNPCC, for example, even with the knowledge that there is a genetic linkage between the disease and markers on chromosome 2, the identification of the gene is unpredictable since the identified markers could be on the order of 9 million base pairs away from the gene of interest. (Peltomaki et al., supra; Marx, Science 260: 751, 1993). The additional observation of genomic instability in HNPCC tumor tissues further complicates identification of that gene.
Even with the present information on prokaryotic mismatch genes and the observation that the products of DNA mismatch repair genes might be involved in genomic instability, it is not clear how to identify eukaryotic homologues of a prokaryotic mismatch repair gene.